Compounds useful as immunomodulators

ABSTRACT

The present disclosure generally relates to compounds useful as immunomodulators. Provided herein are compounds, compositions comprising such compounds, and methods of their use. The disclosure further pertains to pharmaceutical compositions comprising at least one compound according to the disclosure that are useful for the treatment of various diseases, including cancer and infectious diseases.

The present disclosure generally relates to compounds useful asinhibitors of the PD-1/PD-L1 protein/protein and CD80/PD-L1protein/protein interactions. Provided herein are compounds,compositions comprising such compounds, and methods of their use. Thedisclosure further pertains to pharmaceutical compositions comprising atleast one compound according to the disclosure that are useful for thetreatment of various diseases, including cancer and infectious diseases.

Programmed death-1 (CD279) is a receptor on T cells that has been shownto suppress activating signals from the T cell receptor when bound byeither of its ligands, Programmed death-ligand 1 (PD-L1, CD274, B7-H1)or PD-L2 (CD273, B7-DC) (Sharpe et al., Nat. Imm. 2007). When PD-1expressing T cells contact cells expressing its ligands, functionalactivities in response to antigenic stimuli, including proliferation,cytokine secretion, and cytolytic activity are reduced. PD-1/PD-Ligandinteractions down regulate immune responses during resolution of aninfection or tumor, or during the development of self tolerance (KeirMe, Butte M J, Freeman G J, et al. Annu. Rev. Immunol. 2008; 26: Epub).Chronic antigen stimulation, such as that which occurs during tumordisease or chronic infections, results in T cells that express elevatedlevels of PD-1 and are dysfunctional with respect to activity towardsthe chronic antigen (reviewed in Kim and Ahmed, Curr Opin Imm, 2010).This is termed “T cell exhaustion”. B cells also display PD-1/PD-ligandsuppression and “exhaustion”.

PD-L1 has also been shown to interact with CD80 (Butte M J et al.,Immunity 27:111-122 (2007)). The interaction of PD-L1/CD80 on expressingimmune cells has been shown to be an inhibitory one. Blockade of thisinteraction has been shown to abrogate this inhibitory interaction(Paterson A M, et al., J Immunol., 187:1097-1105 (2011); Yang J, et al.J Immunol. August 1; 187(3):1113-9 (2011)).

Blockade of the PD-1/PD-L1 interaction using antibodies to PD-L1 hasbeen shown to restore and augment T cell activation in many systems.Patients with advanced cancer benefit from therapy with a monoclonalantibody to PD-L1 (Brahmer et al., New Engl J Med 2012). Preclinicalanimal models of tumors have shown that blockade of the PD-1/PD-L1pathway by monoclonal antibodies can enhance the immune response andresult in the immune response to a number of histologically distincttumors (Dong H, Chen L. J Mol Med. 2003; 81(5):281-287; Dong H, Strome SE, Salamoa D R, et al. Nat Med. 2002; 8(8):793-800).

Interference with the PD-1/PD-L1 interaction has also shown enhanced Tcell activity in chronic infection systems. Chronic lymphocytic choriomeningitis virus infection of mice also exhibits improved virusclearance and restored immunity with blockade of PD-L1 (Barber D L,Wherry E J, Masopust D, et al. Nature 2006; 439(7077):682-687).Humanized mice infected with HIV-1 show enhanced protection againstviremia and reduced viral depletion of CD4+ T cells (Palmer et al., JImmunol 2013). Blockade of PD-1/PD-L1 through monoclonal antibodies toPD-L1 can restore in vitro antigen-specific functionality to T cellsfrom HIV patients (Day, Nature 2006; Petrovas, J. Exp. Med. 2006;Trautman, Nature Med. 2006; D'Souza, J. Immunol. 2007; Zhang, Blood2007; Kaufmann, Nature Imm. 2007; Kasu, J Immunol. 2010; Porichis, Blood2011), HCV patients [Golden-Mason, J. Virol. 2007; Jeung, J. Leuk. Biol.2007; Urbani, J. Hepatol. 2008; Nakamoto, PLoS Path. 2009; Nakamoto,Gastroenterology 2008] or HBV patients (Boni, J. Virol. 2007; Fisicaro,Gastro. 2010; Fisicaro et al., Gastroenterology, 2012; Boni et al.,Gastro., 2012; Penna et al., J Hep, 2012; Raziorrough, Hepatology 2009;Liang, World J Gastro. 2010; Zhang, Gastro. 2008).

Blockade of the PD-L1/CD80 interaction has also been shown to stimulateimmunity (Yang J., et al., J Immunol. August 1; 187(3):1113-9 (2011)).The immune stimulation resulting from blockade of the PD-L1/CD80interaction has been shown to be enhanced through combination withblockade of further PD-1/PD-L1 or PD-1/PD-L2 interactions.

Alterations in immune cell phenotypes are hypothesized to be animportant factor in septic shock (Hotchkiss, et al., Nat Rev Immunol(2013)). These include increased levels of PD-1 and PD-L1 and T ceollapoptosis (Guignant, et al, Crit. Care (2011)). Antibodies directed toPD-L1 can reduce the level of Immune cell apoptosis (Zhang et al, Crit.Care (2011)). Furthermore, mice lacking PD-1 expression are moreresistant to septic shock symptoms than wildtype mice (Yang J., et al.,J Immunol. August 1; 187(3):1113-9 (2011)). Studies have revealed thatblockade of the interactions of PD-L1 using antibodies can suppressinappropriate immune responses and ameliorate disease symptoms.

In addition to enhancing immunologic responses to chronic antigens,blockade of the PD-1/PD-L1 pathway has also been shown to enhanceresponses to vaccination, including therapeutic vaccination in thecontext of chronic infection (S. J. Ha, S. N. Mueller, E. J. Wherry etal., The Journal of Experimental Medicine, vol. 205, no. 3, pp. 543-555,2008; A. C. Finnefrock, A. Tang, F. Li et al., The Journal ofImmunology, vol. 182, no. 2, pp. 980-98′7, 2009; M.-Y. Song, S.-H. Park,H. J. Nam, D.-H. Choi, and Y.-C. Sung, The Journal of Immunotherapy,vol. 34, no. 3, pp. 297-306, 2011).

The PD-1 pathway is a key inhibitory molecule in T cell exhaustion thatarises from chronic antigen stimulation during chronic infections andtumor disease. Blockade of the PD-1/PD-L1 interaction through targetingthe PD-L1 protein has been shown to restore antigen-specific T cellimmune functions in vitro and in vivo, including enhanced responses tovaccination in the setting of tumor or chronic infection. Accordingly,agents that block the interaction of PD-L1 with either PD-1 or CD80 aredesired.

Applicants found potent compounds that have activity as inhibitors ofthe interaction of PD-L1 with PD-1 and CD80, and thus may be useful fortherapeutic administration to enhance immunity in cancer or infections,including therapeutic vaccine. These compounds are provided to be usefulas pharmaceuticals with desirable stability, bioavailability,therapeutic index, and toxicity values that are important to theirdrugability.

In a first aspect the present disclosure provides a compound of formula(I)

or a pharmaceutically acceptable salt thereof, wherein:

n is 1 or 2;

n′ is 0 or 1;

R¹ is selected from hydrogen and benzyl;

R² is selected from

m is 0, 1, or 2;

Z is —O(CH₂)Ar;

Ar is selected from phenyl and pyridinyl, wherein each ring isoptionally substituted with one substituent selected from C₁-C₄alkoxycarbonyl, C₁-C₄ alkyl, amido, carboxy, cyano, and formyl;

R³ is halo;

R⁴ is —(CH₂)NR⁵R⁶; wherein

R⁵ is selected from hydrogen and C₁-C₄ alkyl;

R⁶ is selected from hydrogen, C₁-C₄ alkyl, and

R⁹ is selected from hydrogen and C₁ alkyl; and

each R^(9′) is independently selected from hydrogen and C₁-C₃ alkyl.

In a first embodiment of the first aspect, the present disclosureprovides a compound of formula (I), or a pharmaceutically acceptablesalt thereof, wherein m is 1 and R³ is halo.

In a second embodiment of the first aspect, the present disclosureprovides a compound of formula (I), or a pharmaceutically acceptablesalt thereof, wherein m is 1 and R³ is halo and R² is

In a third embodiment of the first aspect, the present disclosureprovides a compound of formula (I), or a pharmaceutically acceptablesalt thereof, wherein m is 1 and R³ is halo and R² is

In a fourth embodiment the present disclosure provides a compound offormula (I), or a pharmaceutically acceptable salt thereof, wherein m is1, R³ is halo, and R² is

wherein Z is —O(CH₂)Ar and wherein Ar is pyridinyl optionallysubstituted with one substituent selected from C₁-C₄ alkoxycarbonyl,C₁-C₄alkyl, amido, carboxy, cyano, and formyl.

In a second aspect the present disclosure provides a pharmaceuticalcomposition comprising a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier.

In a third aspect the present disclosure provides a method of enhancing,stimulating, modulating and/or increasing the immune response in asubject in need thereof, said method comprising administering to thesubject a therapeutically effective amount of a compound of formula (I),or a pharmaceutically acceptable salt thereof. In a first embodiment ofthe third aspect the method further comprises administering anadditional agent prior to, after, or simultaneously with the compound offormula (I), or the pharmaceutically acceptable salt thereof. In asecond embodiment the additional agent is an antimicrobial agent, anantiviral agent, a cytotoxic agent, a gene expression modulatory agent,and/or an immune response modifier.

In a fourth aspect the present disclosure provides a method ofinhibiting growth, proliferation, or metastasis of cancer cells in asubject in need thereof, said method comprising administering to thesubject a therapeutically effective amount of a compound of formula (I),or a pharmaceutically acceptable salt. In a first embodiment the canceris selected from melanoma, renal cell carcinoma, squamous non-small celllung cancer (NSCLC), non-squamous NSCLC, colorectal cancer,castration-resistant prostate cancer, ovarian cancer, gastric cancer,hepatocellular carcinoma, pancreatic carcinoma, squamous cell carcinomaof the head and neck, carcinomas of the esophagus, gastrointestinaltract and breast, and a hematological malignancy.

In a fifth aspect the present disclosure provides a method of treatingan infectious disease in a subject in need thereof, the methodcomprising administering to the subject a therapeutically effectiveamount of a compound of formula (I), or a pharmaceutically acceptablesalt thereof. In a first embodiment of the fifth aspect the infectiousdisease is caused by a virus. In a second embodiment the virus isselected from HIV, Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D,herpes viruses, papillomaviruses, and influenza.

In a sixth aspect the present disclosure provides a method of treatingseptic shock in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of acompound of formula (I), or a pharmaceutically acceptable salt thereof.

In a seventh aspect the present disclosure provides a compound offormula (I) or (II) or a pharmaceutically acceptable salt thereof foruse as a medicament.

Unless specifically stated otherwise herein, references made in thesingular may also include the plural. For example, “a” and “an” mayrefer to either one, or one or more.

As used herein, the phase “compound(s) or pharmaceutically acceptablesalts thereof” refers to at least one compound, at least one salt of thecompounds, or a combination thereof. For example, compounds of formula(I) or pharmaceutically acceptable salts thereof includes a compound offormula (I); two compounds of formula (I); a salt of a compound offormula (I); a compound of formula (I) and one or more salts of thecompound of formula (I); and two or more salts of a compound of formula(I).

Unless otherwise indicated, any atom with unsatisfied valences isassumed to have hydrogen atoms sufficient to satisfy the valences.

Throughout the specification, groups and substituents thereof may bechosen by one skilled in the field to provide stable moieties andcompounds.

Listed below are definitions of various terms used to describe thepresent disclosure. These definitions apply to the terms as they areused throughout the specification (unless they are otherwise limited inspecific instances) either individually or as part of a larger group.The definitions set forth herein take precedence over definitions setforth in any patent, patent application, and/or patent applicationpublication incorporated herein by reference.

As used herein, the singular forms “a”, “an”, and “the” include pluralreference unless the context clearly dictates otherwise.

The bicyclic rings of the present disclosure may be monocyclic or fused,spirocyclic, or bridged bicyclic structures.

The term “C₁-C₄alkoxy,” as used herein, refers to a C₁-C₄alkyl groupattached to the parent molecular moiety through an oxygen atom.

The term “C₁-C₄alkoxycarbonyl,” as used herein, refers to a C₁-C₄alkoxygroup attached to the parent molecular moiety through a carbonyl group.

The term “C₁-C₄alkyl,” as used herein, refers to a group derived from astraight or branched chain saturated hydrocarbon containing from one tofour carbon atoms.

The term “amido,” as used herein, refers to —C(O)NH₂.

The term “carbonyl,” as used herein, refers to —C(O)—.

The term “carboxy,” as used herein, refers to —CO₂H.

The term “cyano,” as used herein, refers to —CN.

The term “formyl,” as used herein, refers to —C(O)H.

The terms “halo” and “halogen,” as used herein, refer to F, Cl, Br, orI.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The compounds of formula (I) can form salts which are also within thescope of this disclosure. Unless otherwise indicated, reference to aninventive compound is understood to include reference to one or moresalts thereof. The term “salt(s)” denotes acidic and/or basic saltsformed with inorganic and/or organic acids and bases. In addition, theterm “salt(s) may include zwitterions (inner salts), e.g., when acompound of formula (I) contains both a basic moiety, such as an amineor a pyridine or imidazole ring, and an acidic moiety, such as acarboxylic acid. Pharmaceutically acceptable (i.e., non-toxic,physiologically acceptable) salts are preferred, such as, for example,acceptable metal and amine salts in which the cation does not contributesignificantly to the toxicity or biological activity of the salt.However, other salts may be useful, e.g., in isolation or purificationsteps which may be employed during preparation, and thus, arecontemplated within the scope of the disclosure. Salts of the compoundsof the formula (I) may be formed, for example, by reacting a compound ofthe formula (I) with an amount of acid or base, such as an equivalentamount, in a medium such as one in which the salt precipitates or in anaqueous medium followed by lyophilization.

Exemplary acid addition salts include acetates (such as those formedwith acetic acid or trihaloacetic acid, for example, trifluoroaceticacid), adipates, alginates, ascorbates, aspartates, benzoates,benzenesulfonates, bisulfates, borates, butyrates, citrates,camphorates, camphorsulfonates, cyclopentanepropionates, digluconates,dodecyl sulfates, ethanesulfonates, fumarates, glucoheptanoates,glycerophosphates, hemisulfates, heptanoates, hexanoates, hydrochlorides(formed with hydrochloric acid), hydrobromides (formed with hydrogenbromide), hydroiodides, maleates (formed with maleic acid),2-hydroxyethanesulfonates, lactates, methanesulfonates (formed withmethanesulfonic acid), 2-naphthalenesulfonates, nicotinates, nitrates,oxalates, pectinates, persulfates, 3-phenylpropionates, phosphates,picrates, pivalates, propionates, salicylates, succinates, sulfates(such as those formed with sulfuric acid), sulfonates (such as thosementioned herein), tartrates, thiocyanates, toluenesulfonates such astosylates, undecanoates, and the like.

Exemplary basic salts include ammonium salts, alkali metal salts such assodium, lithium, and potassium salts; alkaline earth metal salts such ascalcium and magnesium salts; barium, zinc, and aluminum salts; saltswith organic bases (for example, organic amines) such as trialkylaminessuch as triethylamine, procaine, dibenzylamine,N-benzyl-β-phenethylamine, 1-ephenamine, N,N′-dib enzylethylene-diamine,dehydroabietylamine, N-ethylpiperidine, benzylamine, dicyclohexylamineor similar pharmaceutically acceptable amines and salts with amino acidssuch as arginine, lysine and the like. Basic nitrogen-containing groupsmay be quaternized with agents such as lower alkyl halides (e.g.,methyl, ethyl, propyl, and butyl chlorides, bromides and iodides),dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, and diamylsulfates), long chain halides (e.g., decyl, lauryl, myristyl and stearylchlorides, bromides and iodides), aralkyl halides (e.g., benzyl andphenethyl bromides), and others. Preferred salts includemonohydrochloride, hydrogensulfate, methanesulfonate, phosphate ornitrate salts.

Various forms of prodrugs are well known in the art and are describedin:

-   a) The Practice of Medicinal Chemistry, Camille G. Wermuth et al.,    Ch 31, (Academic Press, 1996);-   b) Design of Prodrugs, edited by H. Bundgaard, (Elsevier, 1985);-   c) A Textbook of Drug Design and Development, P. Krogsgaard-Larson    and H. Bundgaard, eds. Ch 5, pgs 113-191 (Harwood Academic    Publishers, 1991); and-   d) Hydrolysis in Drug and Prodrug Metabolism, Bernard Testa and    Joachim M. Mayer, (Wiley-VCH, 2003).

In addition, compounds of formula (I), subsequent to their preparation,can be isolated and purified to obtain a composition containing anamount by weight equal to or greater than 99% of a compound of formula(I) (“substantially pure”), which is then used or formulated asdescribed herein. Such “substantially pure” compounds of formula (I) arealso contemplated herein as part of the present disclosure.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent. The present disclosure is intended toembody stable compounds.

“Therapeutically effective amount” is intended to include an amount of acompound of the present disclosure alone or an amount of the combinationof compounds claimed or an amount of a compound of the presentdisclosure in combination with other active ingredients effective toinhibit PD-1/PD-L1 protein/protein and/or CD80/PD-L1 protein/proteininteractions, or effective to treat or prevent cancer or infectiousdisease, such as septic shock, HIV or Hepatitis B, Hepatitis C, andHepatitis D.

As used herein, “treating” or “treatment” cover the treatment of adisease-state in a mammal, particularly in a human, and include: (a)preventing the disease-state from occurring in a mammal, in particular,when such mammal is predisposed to the disease-state but has not yetbeen diagnosed as having it; (b) inhibiting the disease-state, i.e.,arresting its development; and/or (c) relieving the disease-state, i.e.,causing regression of the disease state.

It should be understood that the disclosure encompasses allstereochemical isomeric forms, or mixtures thereof, which possess theability to inhibit the PD-1/PD-L1 protein/protein and/or CD80/PD-L1protein/protein interactions. Individual stereoisomers of compounds canbe prepared synthetically from commercially available starting materialswhich contain chiral centers or by preparation of mixtures ofenantiomeric products followed by separation such as conversion to amixture of diastereomers followed by separation or recrystallization,chromatographic techniques, or direct separation of enantiomers onchiral chromatographic columns. Starting compounds of particularstereochemistry are either commercially available or can be made andresolved by techniques known in the art.

The compounds of the present disclosure are intended to include allisotopes of atoms occurring in the present compounds. Isotopes includethose atoms having the same atomic number but different mass numbers. Byway of general example and without limitation, isotopes of hydrogeninclude deuterium (D) and tritium (T). Isotopes of carbon include ¹³Cand ¹⁴C. Isotopically-labeled compounds of the disclosure can generallybe prepared by conventional techniques known to those skilled in the artor by processes analogous to those described herein, using anappropriate isotopically-labeled reagent in place of the non-labeledreagent otherwise employed. For example, methyl (—CH₃) also includesdeuterated methyl groups such as-CD₃.

Compounds in accordance with formula (I) and/or pharmaceuticallyacceptable salts thereof can be administered by any means suitable forthe condition to be treated, which can depend on the need forsite-specific treatment or quantity of formula (I) compound to bedelivered. Also embraced within this disclosure is a class ofpharmaceutical compositions comprising a compound of formula (I) and/orpharmaceutically acceptable salts thereof; and one or more non-toxic,pharmaceutically-acceptable carriers and/or diluents and/or adjuvants(collectively referred to herein as “carrier” materials) and, ifdesired, other active ingredients. The compounds of formula (I) may beadministered by any suitable route, preferably in the form of apharmaceutical composition adapted to such a route, and in a doseeffective for the treatment intended. The compounds and compositions ofthe present disclosure may, for example, be administered orally,mucosally, rectally, or parentally including intravascularly,intravenously, intraperitoneally, subcutaneously, intramuscularly, andintrasternally in dosage unit formulations containing conventionalpharmaceutically acceptable carriers, adjuvants, and vehicles. Forexample, the pharmaceutical carrier may contain a mixture of mannitol orlactose and microcrystalline cellulose. The mixture may containadditional components such as a lubricating agent, e.g. magnesiumstearate and a disintegrating agent such as crospovidone. The carriermixture may be filled into a gelatin capsule or compressed as a tablet.The pharmaceutical composition may be administered as an oral dosageform or an infusion, for example.

For oral administration, the pharmaceutical composition may be in theform of, for example, a tablet, capsule, liquid capsule, suspension, orliquid. The pharmaceutical composition is preferably made in the form ofa dosage unit containing a particular amount of the active ingredient.For example, the pharmaceutical composition may be provided as a tabletor capsule comprising an amount of active ingredient in the range offrom about 0.1 to 1000 mg, preferably from about 0.25 to 250 mg, andmore preferably from about 0.5 to 100 mg. A suitable daily dose for ahuman or other mammal may vary widely depending on the condition of thepatient and other factors, but, can be determined using routine methods.

Any pharmaceutical composition contemplated herein can, for example, bedelivered orally via any acceptable and suitable oral preparations.Exemplary oral preparations, include, but are not limited to, forexample, tablets, troches, lozenges, aqueous and oily suspensions,dispersible powders or granules, emulsions, hard and soft capsules,liquid capsules, syrups, and elixirs. Pharmaceutical compositionsintended for oral administration can be prepared according to anymethods known in the art for manufacturing pharmaceutical compositionsintended for oral administration. In order to provide pharmaceuticallypalatable preparations, a pharmaceutical composition in accordance withthe disclosure can contain at least one agent selected from sweeteningagents, flavoring agents, coloring agents, demulcents, antioxidants, andpreserving agents.

A tablet can, for example, be prepared by admixing at least one compoundof formula (I) and/or at least one pharmaceutically acceptable saltthereof with at least one non-toxic pharmaceutically acceptableexcipient suitable for the manufacture of tablets. Exemplary excipientsinclude, but are not limited to, for example, inert diluents, such as,for example, calcium carbonate, sodium carbonate, lactose, calciumphosphate, and sodium phosphate; granulating and disintegrating agents,such as, for example, microcrystalline cellulose, sodiumcrosscarmellose, corn starch, and alginic acid; binding agents, such as,for example, starch, gelatin, polyvinyl-pyrrolidone, and acacia; andlubricating agents, such as, for example, magnesium stearate, stearicacid, and talc. Additionally, a tablet can either be uncoated, or coatedby known techniques to either mask the bad taste of an unpleasanttasting drug, or delay disintegration and absorption of the activeingredient in the gastrointestinal tract thereby sustaining the effectsof the active ingredient for a longer period. Exemplary water solubletaste masking materials, include, but are not limited to,hydroxypropyl-methylcellulose and hydroxypropyl-cellulose. Exemplarytime delay materials, include, but are not limited to, ethyl celluloseand cellulose acetate butyrate.

Hard gelatin capsules can, for example, be prepared by mixing at leastone compound of formula (I) and/or at least one salt thereof with atleast one inert solid diluent, such as, for example, calcium carbonate;calcium phosphate; and kaolin.

Soft gelatin capsules can, for example, be prepared by mixing at leastone compound of formula (I) and/or at least one pharmaceuticallyacceptable salt thereof with at least one water soluble carrier, suchas, for example, polyethylene glycol; and at least one oil medium, suchas, for example, peanut oil, liquid paraffin, and olive oil.

An aqueous suspension can be prepared, for example, by admixing at leastone compound of formula (I) and/or at least one pharmaceuticallyacceptable salt thereof with at least one excipient suitable for themanufacture of an aqueous suspension. Exemplary excipients suitable forthe manufacture of an aqueous suspension, include, but are not limitedto, for example, suspending agents, such as, for example, sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethyl-cellulose,sodium alginate, alginic acid, polyvinyl-pyrrolidone, gum tragacanth,and gum acacia; dispersing or wetting agents, such as, for example, anaturally-occurring phosphatide, e.g., lecithin; condensation productsof alkylene oxide with fatty acids, such as, for example,polyoxyethylene stearate; condensation products of ethylene oxide withlong chain aliphatic alcohols, such as, for exampleheptadecaethylene-oxycetanol; condensation products of ethylene oxidewith partial esters derived from fatty acids and hexitol, such as, forexample, polyoxyethylene sorbitol monooleate; and condensation productsof ethylene oxide with partial esters derived from fatty acids andhexitol anhydrides, such as, for example, polyethylene sorbitanmonooleate. An aqueous suspension can also contain at least onepreservative, such as, for example, ethyl and n-propylp-hydroxybenzoate; at least one coloring agent; at least one flavoringagent; and/or at least one sweetening agent, including but not limitedto, for example, sucrose, saccharin, and aspartame.

Oily suspensions can, for example, be prepared by suspending at leastone compound of formula (I) and/or at least one pharmaceuticallyacceptable salt thereof in either a vegetable oil, such as, for example,arachis oil; olive oil; sesame oil; and coconut oil; or in mineral oil,such as, for example, liquid paraffin. An oily suspension can alsocontain at least one thickening agent, such as, for example, beeswax;hard paraffin; and cetyl alcohol. In order to provide a palatable oilysuspension, at least one of the sweetening agents already describedhereinabove, and/or at least one flavoring agent can be added to theoily suspension. An oily suspension can further contain at least onepreservative, including, but not limited to, for example, ananti-oxidant, such as, for example, butylated hydroxyanisol, andalpha-tocopherol.

Dispersible powders and granules can, for example, be prepared byadmixing at least one compound of formula (I) and/or at least onepharmaceutically acceptable salt thereof with at least one dispersingand/or wetting agent; at least one suspending agent; and/or at least onepreservative. Suitable dispersing agents, wetting agents, and suspendingagents are as already described above. Exemplary preservatives include,but are not limited to, for example, anti-oxidants, e.g., ascorbic acid.In addition, dispersible powders and granules can also contain at leastone excipient, including, but not limited to, for example, sweeteningagents; flavoring agents; and coloring agents.

An emulsion of at least one compound of formula (I) and/or at least onepharmaceutically acceptable salt thereof can, for example, be preparedas an oil-in-water emulsion. The oily phase of the emulsions comprisingcompounds of formula (I) may be constituted from known ingredients in aknown manner. The oil phase can be provided by, but is not limited to,for example, a vegetable oil, such as, for example, olive oil andarachis oil; a mineral oil, such as, for example, liquid paraffin; andmixtures thereof. While the phase may comprise merely an emulsifier, itmay comprise a mixture of at least one emulsifier with a fat or an oilor with both a fat and an oil. Suitable emulsifying agents include, butare not limited to, for example, naturally-occurring phosphatides, e.g.,soy bean lecithin; esters or partial esters derived from fatty acids andhexitol anhydrides, such as, for example, sorbitan monooleate; andcondensation products of partial esters with ethylene oxide, such as,for example, polyoxyethylene sorbitan monooleate. Preferably, ahydrophilic emulsifier is included together with a lipophilic emulsifierwhich acts as a stabilizer. It is also preferred to include both an oiland a fat. Together, the emulsifier(s) with or without stabilizer(s)make-up the so-called emulsifying wax, and the wax together with the oiland fat make up the so-called emulsifying ointment base which forms theoily dispersed phase of the cream formulations. An emulsion can alsocontain a sweetening agent, a flavoring agent, a preservative, and/or anantioxidant. Emulsifiers and emulsion stabilizers suitable for use inthe formulation of the present disclosure include Tween 60, Span 80,cetostearyl alcohol, myristyl alcohol, glyceryl monostearate, sodiumlauryl sulfate, glyceryl distearate alone or with a wax, or othermaterials well known in the art.

The compounds of formula (I) and/or at least one pharmaceuticallyacceptable salt thereof can, for example, also be deliveredintravenously, subcutaneously, and/or intramuscularly via anypharmaceutically acceptable and suitable injectable form. Exemplaryinjectable forms include, but are not limited to, for example, sterileaqueous solutions comprising acceptable vehicles and solvents, such as,for example, water, Ringer's solution, and isotonic sodium chloridesolution; sterile oil-in-water microemulsions; and aqueous or oleaginoussuspensions.

Formulations for parenteral administration may be in the form of aqueousor non-aqueous isotonic sterile injection solutions or suspensions.These solutions and suspensions may be prepared from sterile powders orgranules using one or more of the carriers or diluents mentioned for usein the formulations for oral administration or by using other suitabledispersing or wetting agents and suspending agents. The compounds may bedissolved in water, polyethylene glycol, propylene glycol, ethanol, cornoil, cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodiumchloride, tragacanth gum, and/or various buffers. Other adjuvants andmodes of administration are well and widely known in the pharmaceuticalart. The active ingredient may also be administered by injection as acomposition with suitable carriers including saline, dextrose, or water,or with cyclodextrin (i.e. Captisol), cosolvent solubilization (i.e.propylene glycol) or micellar solubilization (i.e. Tween 80).

The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution, and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employed,including synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

A sterile injectable oil-in-water microemulsion can, for example, beprepared by 1) dissolving at least one compound of formula (I) in anoily phase, such as, for example, a mixture of soybean oil and lecithin;2) combining the formula (I) containing oil phase with a water andglycerol mixture; and 3) processing the combination to form amicroemulsion.

A sterile aqueous or oleaginous suspension can be prepared in accordancewith methods already known in the art. For example, a sterile aqueoussolution or suspension can be prepared with a non-toxicparenterally-acceptable diluent or solvent, such as, for example,1,3-butane diol; and a sterile oleaginous suspension can be preparedwith a sterile non-toxic acceptable solvent or suspending medium, suchas, for example, sterile fixed oils, e.g., synthetic mono- ordiglycerides; and fatty acids, such as, for example, oleic acid.

Pharmaceutically acceptable carriers, adjuvants, and vehicles that maybe used in the pharmaceutical compositions of this disclosure include,but are not limited to, ion exchangers, alumina, aluminum stearate,lecithin, self-emulsifying drug delivery systems (SEDDS) such asd-alpha-tocopherol polyethyleneglycol 1000 succinate, surfactants usedin pharmaceutical dosage forms such as Tweens, polyethoxylated castoroil such as CREMOPHOR surfactant (BASF), or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as alpha-, beta-, and gamma-cyclodextrin,or chemically modified derivatives such as hydroxyalkylcyclodextrins,including 2- and 3-hydroxypropyl-cyclodextrins, or other solubilizedderivatives may also be advantageously used to enhance delivery ofcompounds of the formulae described herein.

The pharmaceutically active compounds of this disclosure can beprocessed in accordance with conventional methods of pharmacy to producemedicinal agents for administration to patients, including humans andother mammals. The pharmaceutical compositions may be subjected toconventional pharmaceutical operations such as sterilization and/or maycontain conventional adjuvants, such as preservatives, stabilizers,wetting agents, emulsifiers, buffers etc. Tablets and pills canadditionally be prepared with enteric coatings. Such compositions mayalso comprise adjuvants, such as wetting, sweetening, flavoring, andperfuming agents.

The amounts of compounds that are administered and the dosage regimenfor treating a disease condition with the compounds and/or compositionsof this disclosure depends on a variety of factors, including the age,weight, sex, the medical condition of the subject, the type of disease,the severity of the disease, the route and frequency of administration,and the particular compound employed. Thus, the dosage regimen may varywidely, but can be determined routinely using standard methods. A dailydose of about 0.001 to 100 mg/kg body weight, preferably between about0.0025 and about 50 mg/kg body weight and most preferably between about0.005 to 10 mg/kg body weight, may be appropriate. The daily dose can beadministered in one to four doses per day. Other dosing schedulesinclude one dose per week and one dose per two day cycle.

For therapeutic purposes, the active compounds of this disclosure areordinarily combined with one or more adjuvants appropriate to theindicated route of administration. If administered orally, the compoundsmay be admixed with lactose, sucrose, starch powder, cellulose esters ofalkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesiumstearate, magnesium oxide, sodium and calcium salts of phosphoric andsulfuric acids, gelatin, acacia gum, sodium alginate,polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted orencapsulated for convenient administration. Such capsules or tablets maycontain a controlled-release formulation as may be provided in adispersion of active compound in hydroxypropylmethyl cellulose.

Pharmaceutical compositions of this disclosure comprise at least onecompound of formula (I) and/or at least one pharmaceutically acceptablesalt thereof, and optionally an additional agent selected from anypharmaceutically acceptable carrier, adjuvant, and vehicle. Alternatecompositions of this disclosure comprise a compound of the formula (I)described herein, or a prodrug thereof, and a pharmaceuticallyacceptable carrier, adjuvant, or vehicle.

The compounds of the disclosure inhibit the PD-1/PD-L1 protein/proteinresulting in a PD-L1 blockade. The blockade of PD-L1 can enhance theimmune response to cancerous cells and infectious diseases in mammals,including humans.

In one aspect, the present disclosure relates to treatment of a subjectin vivo using a compound of formula (I) or a salt thereof such thatgrowth of cancerous tumors is inhibited. A compound of formula (I) or asalt thereof may be used alone to inhibit the growth of canceroustumors. Alternatively, a compound of formula (I) or a salt thereof maybe used in conjunction with other immunogenic agents or standard cancertreatments, as described below.

In one embodiment, the disclosure provides a method of inhibiting growthof tumor cells in a subject, comprising administering to the subject atherapeutically effective amount of a compound of formula (I) or a saltthereof.

In one embodiment, a method is provided for treating cancer comprisingadministering to a patient in need thereof, a therapeutically effectiveamount of a compound of formula (I) or a salt thereof. Examples ofcancers include those whose growth may be inhibited using compounds ofthe disclosure include cancers typically responsive to immunotherapy.Non-limiting examples of preferred cancers for treatment includemelanoma (e.g., metastatic malignant melanoma), renal cancer (e.g. clearcell carcinoma), prostate cancer (e.g. hormone refractory prostateadenocarcinoma), breast cancer, colon cancer and lung cancer (e.g.non-small cell lung cancer). Additionally, the disclosure includesrefractory or recurrent malignancies whose growth may be inhibited usingthe compounds of the disclosure.

Examples of other cancers that may be treated using the methods of thedisclosure include bone cancer, pancreatic cancer, skin cancer, cancerof the head or neck, cutaneous or intraocular malignant melanoma,uterine cancer, ovarian cancer, rectal cancer, cancer of the analregion, stomach cancer, testicular cancer, uterine cancer, carcinoma ofthe fallopian tubes, carcinoma of the endometrium, carcinoma of thecervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin'sDisease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of thesmall intestine, cancer of the endocrine system, cancer of the thyroidgland, cancer of the parathyroid gland, cancer of the adrenal gland,sarcoma of soft tissue, cancer of the urethra, cancer of the penis,chronic or acute leukemias including acute myeloid leukemia, chronicmyeloid leukemia, acute lymphoblastic leukemia, chronic lymphocyticleukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of thebladder, cancer of the kidney or urethra, carcinoma of the renal pelvis,neoplasm of the central nervous system (CNS), primary CNS lymphoma,tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitaryadenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer,T-cell lymphoma, environmentally induced cancers including those inducedby asbestos, and combinations of said cancers. The present disclosure isalso useful for treatment of metastatic cancers, especially metastaticcancers that express PD-L1 (Iwai et al. (2005) Int. Immunol.17:133-144).

Optionally, the compounds of formula (I) or salts thereof can becombined with another immunogenic agent, such as cancerous cells,purified tumor antigens (including recombinant proteins, peptides, andcarbohydrate molecules), cells, and cells transfected with genesencoding immune stimulating cytokines (He et al (2004) J. Immunol.173:4919-28). Non-limiting examples of tumor vaccines that can be usedinclude peptides of melanoma antigens, such as peptides of gp100, MAGEantigens, Trp-2, MART1 and/or tyrosinase, or tumor cells transfected toexpress the cytokine GM-CSF.

In humans, some tumors have been shown to be immunogenic such asmelanomas. It is anticipated that by raising the threshold of T cellactivation by PD-L1 blockade, tumor responses are expected to beactivated in the host.

The PD-L1 blockade can be combined with a vaccination protocol. Manyexperimental strategies for vaccination against tumors have been devised(see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCOEducational Book Spring: 60-62; Logothetis, C., 2000, ASCO EducationalBook Spring: 300-302; Khayat, D. 2000, ASCO Educational Book Spring:414-428; Foon, K. 2000, ASCO Educational Book Spring: 730-738; see alsoRestifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp. 3023-3043 inDeVita, V. et al. (eds.), 1997, Cancer: Principles and Practice ofOncology. Fifth Edition). In one of these strategies, a vaccine isprepared using autologous or allogenenic tumor cells. These cellularvaccines have been shown to be most effective when the tumor cells aretransduced to express GM-CSF. GM-CSF has been shown to be a potentactivator of antigen presentation for tumor vaccination (Dranoff et al.(1993) Proc. Natl. Acad. Sci. U.S.A. 90: 3539-43).

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called tumor specificantigens (Rosenberg, S A (1999) Immunity 10: 281-7). In many cases,these tumor specific antigens are differentiation antigens expressed inthe tumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens, and Trp-2. More importantly,many of these antigens can be shown to be the targets of tumor specificT cells found in the host. PD-L1 blockade may be used in conjunctionwith a collection of recombinant proteins and/or peptides expressed in atumor in order to generate an immune response to these proteins. Theseproteins are normally viewed by the immune system as self antigens andare therefore tolerant to them. The tumor antigen may also include theprotein telomerase, which is required for the synthesis of telomeres ofchromosomes and which is expressed in more than 85% of human cancers andin only a limited number of somatic tissues (Kim, N et al. (1994)Science 266: 2011-2013). (These somatic tissues may be protected fromimmune attack by various means). Tumor antigen may also be“neo-antigens” expressed in cancer cells because of somatic mutationsthat alter protein sequence or create fusion proteins between twounrelated sequences (ie. bcr-abl in the Philadelphia chromosome), oridiotype from B cell tumors.

Other tumor vaccines may include the proteins from viruses implicated inhuman cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses(HBV, HDV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). Anotherform of tumor specific antigen which may be used in conjunction withPD-L1 blockade is purified heat shock proteins (HSP) isolated from thetumor tissue itself. These heat shock proteins contain fragments ofproteins from the tumor cells and these HSPs are highly efficient atdelivery to antigen presenting cells for eliciting tumor immunity (Suot,R & Srivastava, P (1995) Science 269:1585-1588; Tamura, Y. et al. (1997)Science 278:117-120).

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle, F. et al. (1998) Nature Medicine 4: 328-332). DCsmay also be transduced by genetic means to express these tumor antigensas well. DCs have also been fused directly to tumor cells for thepurposes of immunization (Kugler, A. et al. (2000) Nature Medicine6:332-336). As a method of vaccination, DC immunization may beeffectively combined with PD-L1 blockade to activate more potentanti-tumor responses.

PD-L1 blockade may also be combined with standard cancer treatments.PD-L1 blockade may be effectively combined with chemotherapeuticregimes. In these instances, it may be possible to reduce the dose ofchemotherapeutic reagent administered (Mokyr, M. et al. (1998) CancerResearch 58: 5301-5304). An example of such a combination is a compoundof this disclosure in combination with dacarbazine for the treatment ofmelanoma. Another example of such a combination is a compound of thisdisclosure in combination with interleukin-2 (IL-2) for the treatment ofmelanoma. The scientific rationale behind the combined use of PD-L1blockade and chemotherapy is that cell death, that is a consequence ofthe cytotoxic action of most chemotherapeutic compounds, should resultin increased levels of tumor antigen in the antigen presentationpathway. Other combination therapies that may result in synergy withPD-L1 blockade through cell death are radiation, surgery, and hormonedeprivation. Each of these protocols creates a source of tumor antigenin the host. Angiogenesis inhibitors may also be combined with PD-L1blockade. Inhibition of angiogenesis leads to tumor cell death which mayfeed tumor antigen into host antigen presentation pathways.

The compounds of this disclosure can also be used in combination withbispecific compounds that target Fc alpha or Fc gammareceptor-expressing effectors cells to tumor cells (see, e.g., U.S. Pat.Nos. 5,922,845 and 5,837,243). Bispecific compounds can be used totarget two separate antigens. For example anti-Fc receptor/anti tumorantigen (e.g., Her-2/neu) bispecific compounds have been used to targetmacrophages to sites of tumor. This targeting may more effectivelyactivate tumor specific responses. The T cell arm of these responseswould be augmented by the use of PD-L1 blockade. Alternatively, antigenmay be delivered directly to DCs by the use of bispecific compoundswhich bind to tumor antigen and a dendritic cell specific cell surfacemarker.

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation of proteinswhich are expressed by the tumors and which are immunosuppressive. Theseinclude among others TGF-beta (Kehrl, J. et al. (1986) J. Exp. Med. 163:1037-1050), IL-10 (Howard, M. & O'Garra, A. (1992) Immunology Today 13:198-200), and Fas ligand (Hahne, M. et al. (1996) Science 274:1363-1365). Inhibitors that bind to and block each of these entities maybe used in combination with the compounds of this disclosure tocounteract the effects of the immunosuppressive agent and favor tumorimmune responses by the host.

Compounds that activate host immune responsiveness can be used incombination with PD-L1 blockade. These include molecules on the surfaceof dendritic cells which activate DC function and antigen presentation.Anti-CD40 compounds are able to substitute effectively for T cell helperactivity (Ridge, J. et al. (1998) Nature 393: 474-478) and can be usedin conjunction with PD-L1 blockade (Ito, N. et al. (2000) Immunobiology201 (5) 527-40). Activating compounds to T cell costimulatory moleculessuch as CTLA-4 (e.g., U.S. Pat. No. 5,811,097), OX-40 (Weinberg, A. etal. (2000) Immunol 164: 2160-2169), 4-1BB (Melero, I. et al. (1997)Nature Medicine 3: 682-685 (1997), and ICOS (Hutloff, A. et al. (1999)Nature 397: 262-266) may also provide for increased levels of T cellactivation.

Bone marrow transplantation is currently being used to treat a varietyof tumors of hematopoietic origin. While graft versus host disease is aconsequence of this treatment, therapeutic benefit may be obtained fromgraft vs. tumor responses. PD-L1 blockade can be used to increase theeffectiveness of the donor engrafted tumor specific T cells.

Other methods of the disclosure are used to treat patients who have beenexposed to particular toxins or pathogens. Accordingly, another aspectof the disclosure provides a method of treating an infectious disease ina subject comprising administering to the subject a therapeuticallyeffective amount of a compound of formula (I) or salts thereof.

Similar to its application to tumors as discussed above, the compound offormula (I) or salts thereof can be used alone, or as an adjuvant, incombination with vaccines, to stimulate the immune response topathogens, toxins, and self-antigens. Examples of pathogens for whichthis therapeutic approach may be particularly useful, include pathogensfor which there is currently no effective vaccine, or pathogens forwhich conventional vaccines are less than completely effective. Theseinclude, but are not limited to HIV, Hepatitis (A, B, C or D),Influenza, Herpes, Giardia, Malaria, Leishmania, Staphylococcus aureus,Pseudomonas Aeruginosa. PD-L1 blockade is particularly useful againstestablished infections by agents such as HIV that present alteredantigens over the course of the infections. These novel epitopes arerecognized as foreign at the time of administration, thus provoking astrong T cell response that is not dampened by negative signals throughPD-1.

Some examples of pathogenic viruses causing infections treatable bymethods of the disclosure include HIV, hepatitis (A, B, C, or D), herpesviruses (e.g., VZV, HSV-1, HAV-6, HSV-2, CMV, and Epstein Barr virus),adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus,coxsackie virus, cornovirus, respiratory syncytial virus, mumps virus,rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus,HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus,rabies virus, JC virus and arboviral encephalitis virus.

Some examples of pathogenic bacteria causing infections treatable bymethods of the disclosure include chlamydia, rickettsial bacteria,mycobacteria, staphylococci, streptococci, pneumonococci, meningococciand conococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax,plague, leptospirosis, and Lymes disease bacteria.

Some examples of pathogenic fungi causing infections treatable bymethods of the disclosure include Candida (albicans, krusei, glabrata,tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus,niger, etc.), Genus Mucorales (mucor, absidia, rhizophus), Sporothrixschenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis,Coccidioides immitis and Histoplasma capsulatum.

Some examples of pathogenic parasites causing infections treatable bymethods of the disclosure include Entamoeba histolytica, Balantidiumcoli, Naegleriafowleri, Acanthamoeba sp., Giardia lambia,Cryptosporidium sp., Pneumocystis carinii, Plasmodium vivax, Babesiamicroti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani,Toxoplasma gondi, and Nippostrongylus brasiliensis.

In all of the above methods, PD-L1 blockade can be combined with otherforms of immunotherapy such as cytokine treatment (e.g., interferons,GM-CSF, G-CSF, IL-2), or bispecific antibody therapy, which provides forenhanced presentation of tumor antigens (see, e.g., Holliger (1993)Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure2:1121-1123), vaccines, or agents that modify gene expression.

The compounds of this disclosure may provoke and amplify autoimmuneresponses. Indeed, induction of anti-tumor responses using tumor celland peptide vaccines reveals that many anti-tumor responses involveanti-self reactivities (depigmentation observed inanti-CTLA-4+GM-CSF-modified B 16 melanoma in van Elsas et al. supra;depigmentation in Trp-2 vaccinated mice (Overwijk, W. et al. (1999)Proc. Natl. Acad. Sci. U.S.A. 96: 2982-2987); autoimmune prostatitisevoked by TRAMP tumor cell vaccines (Hurwitz, A. (2000) supra), melanomapeptide antigen vaccination and vitilago observed in human clinicaltrials (Rosenberg, S A and White, D E (1996) J. Immunother EmphasisTumor Immunol 19 (1): 81-4).

Therefore, it is possible to consider using anti-PD-L1 blockade inconjunction with various self proteins in order to devise vaccinationprotocols to efficiently generate immune responses against these selfproteins for disease treatment. For example, Alzheimer's diseaseinvolves inappropriate accumulation of A.beta.peptide in amyloiddeposits in the brain; antibody responses against amyloid are able toclear these amyloid deposits (Schenk et al., (1999) Nature 400:173-177).

Other self proteins may also be used as targets such as IgE for thetreatment of allergy and asthma, and TNF.alpha. for rheumatoidarthritis. Finally, antibody responses to various hormones may beinduced by the use of a compound of formula (I) or salts thereof.Neutralizing antibody responses to reproductive hormones may be used forcontraception. Neutralizing antibody response to hormones and othersoluble factors that are required for the growth of particular tumorsmay also be considered as possible vaccination targets.

Analogous methods as described above for the use of anti-PD-L1 antibodycan be used for induction of therapeutic autoimmune responses to treatpatients having an inappropriate accumulation of other self-antigens,such as amyloid deposits, including A.beta. in Alzheimer's disease,cytokines such as TNF alpha, and IgE.

The compounds of this disclosure may be used to stimulateantigen-specific immune responses by co-administration of a compound offormula (I) or salts thereof with an antigen of interest (e.g., avaccine). Accordingly, in another aspect the disclosure provides amethod of enhancing an immune response to an antigen in a subject,comprising administering to the subject: (i) the antigen; and (ii) acompound of formula (I) or salts thereof, such that an immune responseto the antigen in the subject is enhanced. The antigen can be, forexample, a tumor antigen, a viral antigen, a bacterial antigen or anantigen from a pathogen. Non-limiting examples of such antigens includethose discussed in the sections above, such as the tumor antigens (ortumor vaccines) discussed above, or antigens from the viruses, bacteriaor other pathogens described above.

As previously described, the compounds of the disclosure can beco-administered with one or more other therapeutic agents, e.g., acytotoxic agent, a radiotoxic agent or an immunosuppressive agent. Thecompounds of the disclosure can be administered before, after orconcurrently with the other therapeutic agent or can be co-administeredwith other known therapies, e.g., an anti-cancer therapy, e.g.,radiation. Such therapeutic agents include, among others,anti-neoplastic agents such as doxorubicin (adriamycin), cisplatinbleomycin sulfate, carmustine, chlorambucil, decarbazine andcyclophosphamide hydroxyurea which, by themselves, are only effective atlevels which are toxic or subtoxic to a patient. Cisplatin isintravenously administered as a 100 mg/dose once every four weeks andadriamycin is intravenously administered as a 60-75 mg/mL dose onceevery 21 days. Co-administration of a compound of formula (I) or saltsthereof, with chemotherapeutic agents provides two anti-cancer agentswhich operate via different mechanisms which yield a cytotoxic effect tohuman tumor cells. Such co-administration can solve problems due todevelopment of resistance to drugs or a change in the antigenicity ofthe tumor cells which would render them unreactive with the antibody.

Also within the scope of the present disclosure are kits comprising acompound of formula (I) or salts thereof and instructions for use. Thekit can further contain at least one additional reagent. Kits typicallyinclude a label indicating the intended use of the contents of the kit.The term label includes any writing, or recorded material supplied on orwith the kit, or which otherwise accompanies the kit.

The above other therapeutic agents, when employed in combination withthe compounds of the present disclosure, may be used, for example, inthose amounts indicated in the Physicians' Desk Reference (PDR) or asotherwise determined by one of ordinary skill in the art. In the methodsof the present disclosure, such other therapeutic agent(s) may beadministered prior to, simultaneously with, or following theadministration of the inventive compounds.

In one embodiment, the compounds of formula (I) inhibit the PD-1/PD-L1interaction with IC₅₀ values of 20 μM or less, for example, from 0.48 to20 μM, as measured by the PD-1/PD-L1 Homogenous Time-ResolvedFluorescence (HTRF) binding assay.

EXAMPLES

The invention is further defined in the following Examples. It should beunderstood that the Examples are given by way of illustration only. Fromthe above discussion and the Examples, one skilled in the art canascertain the essential characteristics of the invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications to adapt the invention to various uses and conditions.As a result, the invention is not limited by the illustrative examplesset forth hereinbelow, but rather is defined by the claims appendedhereto.

As used in the present specification, the following terms have themeanings indicated: DMF for N,N-dimethylformamide, MeOH for methanol,HATU for(1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate), TFA for trifluoroacetic acid, DCM fordichloromethane, DMSO for dimethylsulfoxide, THF for tetrahydrofuran, rtor RT or Rt for room temperature or retention time (context willdictate), and h or hr or hrs for hours

Intermediate: (R)-ethyl 4-(3-(benzyloxy)pyrrolidin-1-yl)butanoate

A mixture of ethyl 4-chlorobutanoate (176 mg, 1.170 mmol),(R)-3-(benzyloxy)pyrrolidine, HCl (250 mg, 1.170 mmol), and K₂CO₃ (647mg, 4.68 mmol) in DMF (4 mL) was stirred at 50° C. for 16 hrs. Thesolvent was removed, and residue was purified by silic gel column(biotage 25s, 5% NH₃ in MeOH/CH₂Cl₂=0 to 10%) to give 118 mg (55%) oftarget compound. ¹H NMR (500 MHz, CHLOROFORM-d) δ 7.39-7.32 (m, 4H),7.32-7.26 (m, 1H), 4.54-4.45 (m, 2H), 4.19-4.10 (m, 3H), 2.82 (dd,J=10.0, 6.2 Hz, 1H), 2.72-2.65 (m, 1H), 2.62 (dd, J=10.0, 3.7 Hz, 1H),2.57-2.44 (m, 3H), 2.36 (t, J=7.4 Hz, 2H), 2.14-2.05 (m, 1H), 1.94-1.80(m, 3H), 1.27 (t, J=6.8 Hz, 3H).

Intermediate: (R)-4-(3-(benzyloxy)pyrrolidin-1-yl)butanoic acid, lithiumsalt

A solution of lithium hydroxide (1M, 2.58 mL, 2.58 mmol) was added to asolution of (R)-ethyl 4-(3-(benzyloxy)pyrrolidin-1-yl)butanoate (188 mg,0.645 mmol) in methanol (4 mL) and THF (4 mL). The resulted solution wasstirred at room temperature overnight. The solvent was removed. Theresidue (240 mg) was used directly for the next step without furtherpurification.

Example 1001:1,1′-(2,8-diazaspiro[5.5]undecane-2,8-diyl)bis(4-((R)-3-(benzyloxy)pyrrolidin-1-yl)butan-1-one)

To a screw capped vial was added 2,8-diazaspiro[5.5]undecane, 2 TFA (60mg, 0.157 mmol, crude), (R)-4-(3-(benzyloxy)pyrrolidin-1-yl)butanoicacid, lithium salt (127 mg, 0.471 mmol, crude) in DMF (2 mL) along withN,N-diisopropylethylamine (0.164 mL, 0.942 mmol) and HATU(2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethylisouroniumhexafluorophosphate) (185 mg, 0.487 mmol). The mixture was stirred atroom temperature for 2 days. The crude material was purified viapreparative LC/MS with the following conditions: Column: Sun fire C1819×150 mm; Mobile Phase A: 5:95 acetonitrile: water with 0.1% TFA;Mobile Phase B: 95:5 acetonitrile: water with 0.1% TFA; Gradient: 40-80%B over 25 minutes, then a 5-minute hold at 100% B; Flow: 25 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation to give target compound (43 mg, 43%). ¹H NMR(500 MHz, CHLOROFORM-d) δ 7.41-7.29 (m, 10H), 4.64-4.46 (m, 4H),4.43-4.28 (m, 2H), 4.02-3.72 (m, 4H), 3.47-2.77 (m, 12H), 2.73-2.14 (m,14H), 2.11-1.90 (m, 2H), 1.64 (d, J=12.9 Hz, 6H), 1.48-1.26 (m, 2H).

Example 1002:1,1′-(2,8-diazaspiro[5.5]undecane-2,8-diyl)bis(4-((R)-3-hydroxypyrrolidin-1-yl)butan-1-one)

A mixture of Pd/C (28.4 mg, 1.334 μmol) and1,1′-(2,8-diazaspiro[5.5]undecane-2,8-diyl)bis(4-((R)-3-(benzyloxy)pyrrolidin-1-yl)butan-1-one)(43 mg, 0.067 mmol) in MeOH (5 mL) was stirred at rt under a balloon ofhydrogen for 20 hrs. The reaction mixture was diluted with MeOH (20 mL).The mixture was filtered and the solvent removed. The crude material waspurified via preparative LC/MS with the following conditions: Column:XBridge C18, 19×200 mm, 5-μm particles; Mobile Phase A: 5:95acetonitrile: water with 10-mM ammonium acetate; Mobile Phase B: 95:5acetonitrile: water with 10-mM ammonium acetate; Gradient: 5-40% B over15 minutes, then a 5-minute hold at 100% B; Flow: 20 mL/min. Fractionscontaining the desired product were combined and dried via centrifugalevaporation. The yield of the product was 4.8 mg (15%), and itsestimated purity by LCMS analysis was 100%.

Two analytical LC/MS injections were used to determine the final purity.Injection 1 conditions: Column: Waters BEH C18, 2.0×50 mm, 1.7-μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 10 mM ammoniumacetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mM ammoniumacetate; Temperature: 50° C.; Gradient: 0% B, 0-100% B over 3 minutes,then a 0.75-minute hold at 100% B; Flow: 1 mL/min; Detection: UV at 220nm. Injection 2 conditions: Column: Waters BEH C18, 2.0×50 mm, 1.7 μmparticles; Mobile Phase A: 5:95 acetonitrile:water with 0.1%trifluoroacetic acid; Mobile Phase B: 95:5 acetonitrile:water with 0.1%trifluoroacetic acid; Temperature: 50° C.; Gradient: 0% B, 0-100% B over3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.0 mL/min;Detection: UV at 220 nm. LCMS (Injection 1 condition) Rt (Retentiontime)=0.527 min, ESI m/z 465 (M+1). LCMS (Injection 2 condition)Rt=0.689 min, ESI m/z 465 (M+1). ¹H NMR (500 MHz, DMSO-d₆) δ 4.18 (br.s., 2H), 3.11-3.04 (m, 1H), 2.99-2.90 (m, 1H), 2.70 (m, 2H), 2.57 (m,2H), 2.48-2.15 (m, 16H), 1.97 (dd, J=13.0, 7.5 Hz, 2H), 1.70-1.58 (m,4H), 1.58-1.32 (m, 12H), 1.24 (br. s., 2H).

Intermediate: tert-butyl8-(4-((R)-3-(benzyloxy)pyrrolidin-1-yl)butanoyl)-2,8-diazaspiro[5.5]undecane-2-carboxylate

To a screw capped vial was added tert-butyl2,8-diazaspiro[5.5]undecane-2-carboxylate (218 mg, 0.858 mmol),(R)-4-(3-(benzyloxy)pyrrolidin-1-yl)butanoic acid, lithium salt (255 mg,0.944 mmol) (crude) in DMF (4 mL) along with N,N-diisopropylethylamine(0.448 mL, 2.57 mmol) and HATU (652 mg, 1.716 mmol). The mixture wasstirred at room temperature for 20 hrs. The crude material was purifiedvia preparative LC/MS with the following conditions: Column: Sunfire C1819×150 mm; Mobile Phase A: 5:95 acetonitrile: water with 0.1% TFA;Mobile Phase B: 95:5 acetonitrile: water with 0.1% TFA; Gradient: 40-80%B over 25 minutes, then a 5-minute hold at 100% B; Flow: 25 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation to give 91 mg (20%) of target compound. ¹N NMR(500 MHz, CHLOROFORM-d) δ 7.44-7.29 (m, 5H), 4.62-4.30 (m, 4H),4.04-3.69 (m, 3H), 3.61-2.92 (m, 11H), 2.82-2.40 (m, 3H), 2.36-1.97 (m,5H), 1.90-1.30 (m, 5H), 1.46 (s, 9H).

Intermediate:4-((R)-3-(benzyloxy)pyrrolidin-1-yl)-1-(2,8-diazaspiro[5.5]undecan-2-yl)butan-1-one

Trifluoroacetic acid (0.084 mL, 1.093 mmol) was added to a solution oftert-butyl8-(4-((R)-3-(benzyloxy)pyrrolidin-1-yl)butanoyl)-2,8-diazaspiro[5.5]undecane-2-carboxylate(91 mg, 0.182 mmol) in DCM (3 mL) at 0° C. and the reaction mixture wasstirred at rt for 20 hrs. The solvent was removed, and the crude productwas used directly for the next reaction without further purification.

Intermediate: tert-butyl3-(2-chloro-5-((5-cyanopyridin-3-yl)methoxy)-4-formylphenyl)propanoate

A mixture of 5-((5-bromo-4-chloro-2-formylphenoxy)methyl)nicotinonitrile(564 mg, 1.605 mmol) (crude, prepared in two steps from3-bromo-4-chlorophenol via formylation and then alkylation with5-(chloromethyl)nicotinonitrile), potassium(3-(tert-butoxy)-3-oxopropyl)trifluoroborate (568 mg, 2.408 mmol),cesium carbonate (2353 mg, 7.22 mmol), and[1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (176 mg,0.241 mmol) in toluene (12.5 mL) and Water (3.75 mL) was flushed withN₂, and then heated to 85° C. for 5 hrs. The solvent was removed. Thecrude material was purified via preparative LC/MS with the followingconditions: Column: Waters-Sunfire, 30×100 mm, 5-μm particles; MobilePhase A: 90% Water-10% acetonitile-0.1% Trifluoroacetic acid; MobilePhase B: 90% acetonitrile-10% water-0.1% Trifluoroacetic acid; Gradient:20-100% B over 20 minutes, then a 2-minute hold at 100% B; Flow: 40mL/min.; 57 mg (8.9%) of the target compound was obtained. ¹H NMR (500MHz, CHLOROFORM-d) δ 10.39 (s, 1H), 8.98 (br. s., 2H), 8.21 (br. s.,1H), 7.87 (s, 1H), 7.07 (s, 1H), 5.28 (s, 2H), 3.08 (t, J=7.3 Hz, 2H),2.63 (t, J=7.3 Hz, 2H), 1.45 (s, 9H).

Intermediate:3-(2-chloro-5-((5-cyanopyridin-3-yl)methoxy)-4-formylphenyl)propanoicacid

A lithium hydroxide solution (1M, 0.569 mL, 0.569 mmol) was added to asolution of tert-butyl3-(2-chloro-5-((5-cyanopyridin-3-yl)methoxy)-4-formylphenyl)propanoate(57 mg, 0.142 mmol) in MeOH (1 mL) and THF (1 mL). The resultingsolution was stirred at rt overnight. The solvent was removed, and theresidue was used directly for the next step reaction without furtherpurification. The LCMS showed the3-(2-chloro-5-((5-cyanopyridin-3-yl)methoxy)-4-formylphenyl)propanoicacid product and the presence of3-(2-chloro-4-formyl-5-((5-(imino(methoxy)methyl)pyridin-3-yl)methoxy)phenyl)propanoicacid.

Intermediate:5-((5-(3-(8-(4-((R)-3-(benzyloxy)pyrrolidin-1-yl)butanoyl)-2,8-diazaspiro[5.5]undecan-2-yl)-3-oxopropyl)-4-chloro-2-formylphenoxy)methyl)nicotinonitrile

To a screw capped vial was added3-(2-chloro-5-((5-cyanopyridin-3-yl)methoxy)-4-(hydroxy(methoxy)methyl)phenyl)propanoicacid (53.5 mg, 0.142 mmol),4-((R)-3-(benzyloxy)pyrrolidin-1-yl)-1-(2,8-diazaspiro[5.5]undecan-2-yl)butan-1-one(56.7 mg, 0.142 mmol) in DMF (2 mL) along with N,N-diisopropylethylamine(0.074 mL, 0.426 mmol) and HATU (108 mg, 0.284 mmol). The mixture wasstirred at room temperature for 20 hrs. The crude material was purifiedvia preparative LC/MS with the following conditions: Column: Sunfire C1819×150 mm; Mobile Phase A: 5:95 acetonitrile: water with 0.1% TFA;Mobile Phase B: 95:5 acetonitrile: water with 0.1% TFA; Gradient: 40-80%B over 25 minutes, then a 5-minute hold at 100% B; Flow: 25 mL/min.Fractions containing the desired product were combined and dried viacentrifugal evaporation (27 mg). The LCMS showed the product5-((5-(3-(8-(4-((R)-3-(benzyloxy)pyrrolidin-1-yl)butanoyl)-2,8-diazaspiro[5.5]undecan-2-yl)-3-oxopropyl)-4-chloro-2-formylphenoxy)methyl)nicotinonitrileand the presence of methyl5-((5-(3-(8-(4-((R)-3-(benzyloxy)pyrrolidin-1-yl)butanoyl)-2,8-diazaspiro[5.5]undecan-2-yl)-3-oxopropyl)-4-chloro-2-formylphenoxy)methyl)nicotinimidate.

Intermediate:5-((5-(3-(8-(4-((R)-3-(benzyloxy)pyrrolidin-1-yl)butanoyl)-2,8-diazaspiro[5.5]undecan-2-yl)-3-oxopropyl)-4-chloro-2-formylphenoxy)methyl)nicotinamide

5-((5-(3-(8-(4-((R)-3-(benzyloxy)pyrrolidin-1-yl)butanoyl)-2,8-diazaspiro[5.5]undecan-2-yl)-3-oxopropyl)-4-chloro-2-formylphenoxy)methyl)nicotinamide(18 mg) was also isolated from the amide coupling reaction mixtureabove. ¹H NMR (500 MHz, CHLOROFORM-d) δ 10.41-10.29 (m, 1H), 9.28 (br.s., 1H), 9.03 (br. s., 1H), 8.94-8.80 (m, 1H), 7.85-7.73 (m, 1H),7.42-7.20 (m, 6H), 5.49-5.34 (m, 2H), 4.62-4.43 (m, 2H), 4.34 (br. s.,1H), 4.02-3.79 (m, 2H), 3.76-3.39 (m, 2H), 3.38-2.40 (m, 12H), 2.38-1.91(m, 4H), 1.76-1.12 (m, 12H).

Example 1003:(2S)-2-((4-(3-(8-(4-((R)-3-(benzyloxy)pyrrolidin-1-yl)butanoyl)-2,8-diazaspiro[5.5]undecan-2-yl)-3-oxopropyl)-2-((5-carbamoylpyridin-3-yl)methoxy)-5-chlorobenzyl)amino)-3-hydroxy-2-methylpropanoicacid

A mixture of5-((5-(3-(8-(4-((R)-3-(benzyloxy)pyrrolidin-1-yl)butanoyl)-2,8-diazaspiro[5.5]undecan-2-yl)-3-oxopropyl)-4-chloro-2-formylphenoxy)methyl)nicotinamide(18 mg, 0.024 mmol) and (S)-2-amino-3-hydroxy-2-methylpropanoic acid(8.64 mg, 0.073 mmol), and sodium triacetoxyborohydride (15.89 mg, 0.075mmol) in DMF (1 mL) was stirred at room temperature overnight. Thesolvent was removed. The crude material was purified via preparativeLC/MS with the following conditions: Column: XBridge C18, 19×200 mm,5-μm particles; Mobile Phase A: 5:95 acetonitrile: water with 10-mMammonium acetate; Mobile Phase B: 95:5 acetonitrile: water with 10-mMammonium acetate; Gradient: 10-50% B over 20 minutes, then a 5-minutehold at 100% B; Flow: 20 mL/min. Fractions containing the desiredproduct were combined and dried via centrifugal evaporation. The yieldof the product was 5.6 mg (27%).

Two analytical LC/MS injections were used to determine the final purity.Injection 1 conditions: Column: Waters Acquity UPLC BEH C18, 2.1×50 mm,1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10 mMammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mMammonium acetate; Temperature: 50° C.; Gradient: 0-100% B over 3minutes, then a 0.75-minute hold at 100% B; Flow: 1.0 mL/min; Detection:UV at 220 nm. Injection 2 conditions: Column: Waters Acquity UPLC BEHC18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5acetonitrile:water with 0.1% trifluoroacetic acid; Temperature: 50° C.;Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B;Flow: 1.0 mL/min; Detection: UV at 220 nm. LCMS (Injection 1 condition)Rt=1.215 min, ESI m/z 847 (M+1), m/z 845 (M−1). LCMS (Injection 2condition) Rt=1.308 min, ESI m/z 847 (M+1), m/z 845 (M−1). ¹H NMR (500MHz, DMSO-d₆) δ 9.26 (br. s., 1H), 8.97 (s, 1H), 8.78 (s, 1H), 8.64 (br.s., 1H), 7.56-7.45 (m, 1H), 7.41-7.17 (m, 7H), 5.26 (br. s., 2H), 4.40(d, J=12.1 Hz, 2H), 4.20-3.96 (m, 3H), 3.62-3.50 (m, 4H), 3.48-3.11 (m,4H), 2.93 (br. s., 3H), 2.65 (br. s., 5H), 2.44-2.20 (m, 6H), 2.09-1.93(m, 1H), 1.77-1.31 (m, 10H), 1.29-1.19 (m, 1H), 1.26 (s, 3H).

Example 1004:(2S)-2-((4-(3-(8-(4-((R)-3-(benzyloxy)pyrrolidin-1-yl)butanoyl)-2,8-diazaspiro[5.5]undecan-2-yl)-3-oxopropyl)-5-chloro-2-((5-cyanopyridin-3-yl)methoxy)benzyl)amino)-3-hydroxy-2-methylpropanoicacid

A mixture of5-((5-(3-(8-(4-((R)-3-(benzyloxy)pyrrolidin-1-yl)butanoyl)-2,8-diazaspiro[5.5]undecan-2-yl)-3-oxopropyl)-4-chloro-2-formylphenoxy)methyl)nicotinonitrile(27 mg, as prepared above) and (S)-2-amino-3-hydroxy-2-methylpropanoicacid (12.72 mg, 0.107 mmol), and sodium triacetoxyborohydride (23.39 mg,0.110 mmol) in DMF (1 mL) was stirred at room temperature overnight. Thesolvent was removed. The crude material was purified via preparativeLC/MS with the following conditions: Column: XBridge C18, 19×200 mm,5-μm particles; Mobile Phase A: 5:95 methanol: water with 10-mM ammoniumacetate; Mobile Phase B: 95:5 methanol: water with 10-mM ammoniumacetate; Gradient: 40-100% B over 25 minutes, then a 8-minute hold at100% B; Flow: 20 mL/min. Fractions containing the desired product werecombined and dried via centrifugal evaporation.

Two analytical LC/MS injections were used to determine the final purity.Injection 1 conditions: Column: Waters Acquity UPLC BEH C18, 2.1×50 mm,1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:water with 10 mMammonium acetate; Mobile Phase B: 95:5 acetonitrile:water with 10 mMammonium acetate; Temperature: 50° C.; Gradient: 0-100% B over 3minutes, then a 0.75-minute hold at 100% B; Flow: 1.0 mL/min; Detection:UV at 220 nm. Injection 2 conditions: Column: Waters Acquity UPLC BEHC18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5acetonitrile:water with 0.1% trifluoroacetic acid; Temperature: 50° C.;Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B;Flow: 1.0 mL/min; Detection: UV at 220 nm. LCMS (Injection 1 condition)Rt=1.326 min, m/z 829 (M+1), m/z 827 (M−1). LCMS (Injection 2 condition)Rt=1.332 min, m/z 829 (M+1). ¹H NMR (500 MHz, DMSO-d₆) δ 9.00 (s, 2H),8.46 (br. s., 1H), 7.54-7.46 (m, 1H), 7.41-7.22 (m, 5H), 7.14 (d, J=16.5Hz, 1H), 5.26 (br. s., 2H), 4.41 (d, J=10.6 Hz, 2H), 4.15-4.02 (m, 1H),3.91 (br. s., 2H), 3.63-3.47 (m, 1H), 3.4-3.1 (m, 5H), 2.90 (br. s.,3H), 2.61 (br. s., 5H), 2.45-2.16 (m, 6H), 2.09-1.94 (m, 1H), 1.81-1.29(m, 12H), 1.29-1.17 (m, 1H), 1.22 (s, 3H).

Example 1005:(2S)-2-((4-(3-(8-(4-((R)-3-(benzyloxy)pyrrolidin-1-yl)butanoyl)-2,8-diazaspiro[5.5]undecan-2-yl)-3-oxopropyl)-5-chloro-2-((5-(methoxycarbonyl)pyridin-3-yl)methoxy)benzyl)amino)-3-hydroxy-2-methylpropanoicacid

Example 1005 was also isolated from the reaction mixture for Example1004. Two analytical LC/MS injections were used to determine the finalpurity. Injection 1 conditions: Column: Waters Acquity UPLC BEH C18,2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95 acetonitrile:waterwith 10 mM ammonium acetate; Mobile Phase B: 95:5 acetonitrile:waterwith 10 mM ammonium acetate; Temperature: 50° C.; Gradient: 0-100% Bover 3 minutes, then a 0.75-minute hold at 100% B; Flow: 1.0 mL/min;Detection: UV at 220 nm. Injection 2 conditions: Column: Waters AcquityUPLC BEH C18, 2.1×50 mm, 1.7-μm particles; Mobile Phase A: 5:95acetonitrile:water with 0.1% trifluoroacetic acid; Mobile Phase B: 95:5acetonitrile:water with 0.1% trifluoroacetic acid; Temperature: 50° C.;Gradient: 0-100% B over 3 minutes, then a 0.75-minute hold at 100% B;Flow: 1.0 mL/min; Detection: UV at 220 nm. LCMS (Injection 1 condition)Rt=1.344 min, ESI m/z 862 (M+1), m/z 860 (M−1). LCMS (Injection 2condition) Rt=1.314 min, ESI m/z 862 (M+1). ¹H NMR (500 MHz, DMSO-d₆) δ9.05 (s, 1H), 8.96 (s, 1H), 8.41 (br. s., 1H), 7.54-7.47 (m, 1H),7.38-7.23 (m, 5H), 7.20-7.12 (m, 1H), 5.29 (br. s., 2H), 4.41 (d, J=12.1Hz, 2H), 4.13-4.00 (m, 1H), 3.94-3.84 (m, 2H), 3.90 (s, 3H), 3.53 (d,J=18.7 Hz, 1H), 3.4-3.1 (m, 5H), 2.90 (m, 3H), 2.75-2.48 (br. s., 5H),2.46-2.15 (m, 6H), 2.09-1.93 (m, 1H), 1.79-1.29 (m, 12H), 1.28-1.17 (m,1H), 1.20 (s, 3H).

BIOLOGICAL ASSAY

The ability of the compounds of formula (I) to bind to PD-L1 wasinvestigated using a PD-1/PD-L1 Homogenous Time-Resolved Fluorescence(HTRF) binding assay.

1. Homogenous Time-Resolved Fluorescence (HTRF) Binding Assay.

The interaction of PD-1 and PD-L1 can be assessed using soluble,purified preparations of the extracellular domains of the two proteins.The PD-1 and PD-L1 protein extracellular domains were expressed asfusion proteins with detection tags, for PD-1, the tag was the Fcportion of Immunoglobulin (PD-1-Ig) and for PD-L1 it was the 6 histidinemotif (PD-L1-His). All binding studies were performed in an HTRF assaybuffer consisting of dPBS supplemented with 0.1% (with) bovine serumalbumin and 0.05% (v/v) Tween-20. For the h/PD-L1-His binding assay,inhibitors were pre-incubated with PD-L1-His (10 nM final) for 15 m in 4μl of assay buffer, followed by addition of PD-1-Ig (20 nM final) in 1μl of assay buffer and further incubation for 15 m. HTRF detection wasachieved using europium crypate-labeled anti-Ig (1 nM final) andallophycocyanin (APC) labeled anti-His (20 nM final). Antibodies werediluted in HTRF detection buffer and 5 μl was dispensed on top of thebinding reaction. The reaction mixture was allowed to equilibrate for 30minutes and the resulting signal (665 nm/620 nm ratio) was obtainedusing an EnVision fluorometer. Additional binding assays wereestablished between the human proteins PD-1-Ig/PD-L2-His (20 & 5 nM,respectively) and CD80-His/PD-L1-Ig (100 & 10 nM, respectively).

Recombinant Proteins: Human PD-1 (25-167) with a C-terminal human Fcdomain of immunoglobulin G (Ig) epitope tag [hPD-1 (25-167)-3S-IG] andhuman PD-L1 (18-239) with a C-terminal His epitope tag [hPD-L1(18-239)-TVMV-His] were expressed in HEK293T cells and purifiedsequentially by ProteinA affinity chromatography and size exclusionchromatography. Human PD-L2-His and CD80-His was obtained throughcommercial sources.

Sequence of recombinant human PD-1-Ig hPD1(25-167)-3S-IG 1LDSPDRPWNP PTFSPALLVV TEGDNATFTC SFSNTSESFV LNWYRMSPSN 51QTDKLAAFPE DRSQPGQDCR FRVTQLPNGR DFHMSVVRAR RNDSGTYLCG 101AISLAPKAQI KESLRAELRV TERRAEVPTA HPSPSPRPAG QFQGSPGGGG 151GREPKSSDKT RTSPPSPAPE LLGGSSVFLF PPKPKDTLMI SRTPEVTCVV 201VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW 251LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP SRDELTKNQV 301SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD 351KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK (SEQ ID NO: 1)Sequence of recombinant human PD-Li-His hPDL1(18-239)-TVMV-His 1AFTVTVPKDL YVVEYGSNMT IECKFPVEKQ LDLAALIVYW EMEDKNIIQF 51VHGEEDLKVQ HSSYRQRARL LKDQLSLGNA ALQITDVKLQ DAGVYRCMIS 101YGGADYKRIT VKVNAPYNKI NQRILVVDPV TSEHELTCQA EGYPKAEVIW 151TSSDHQVLSG KTTTTNSKRE EKLFNVTSTL RINTTTNEIF YCTFRRLDPE 201ENHTAELVIP ELPLAHPPNE RTGSSETVRF QGHHHHHH (SEQ ID NO: 2)

The table below lists the IC₅₀ values for representative examples ofthis disclosure measured in the PD-1/PD-L1 Homogenous Time-ResolvedFluorescence (HTRF) binding assay.

Example Number HTRF IC50 (μM) Example 1001 >2.50 Example 1002 0.84Example 1003 >10.00 Example 1004 >10.00 Example 1005 >10.00

The compounds of formula (I) possess activity as inhibitors of thePD-1/PD-L1 interaction, and therefore, may be used in the treatment ofdiseases or deficiencies associated with the PD-1/PD-L1 interaction. Viainhibition of the PD-1/PD-L1 interaction, the compounds of the presentdisclosure may be employed to treat infectious diseases such as HIV,septic shock, Hepatitis A, B, C, or D and cancer.

What is claimed is:
 1. A compound of formula (I):

or a pharmaceutically acceptable salt thereof, wherein: n is 1 or 2; n′is 0 or 1; R¹ is selected from hydrogen and benzyl; R² is selected from

m is 0, 1, or 2; Z is —O(CH₂)Ar; Ar is selected from phenyl andpyridinyl, wherein each ring is optionally substituted with onesubstituent selected from C₁-C₄alkoxycarbonyl, C₁-C₄alkyl, amido,carboxy, cyano, and formyl; R³ is halo; R⁴ is —(CH₂)NR⁵R⁶; wherein R⁵ isselected from hydrogen and C₁-C₄alkyl;

R⁶ is selected from hydrogen, C₁-C₄alkyl, and R⁹ is selected fromhydrogen and C₁alkyl; and each R^(9′) is independently selected fromhydrogen and C₁-C₃alkyl.
 2. A compound of claim 1, or a pharmaceuticallyacceptable salt thereof, wherein m is 1 and R³ is halo.
 3. A compound ofclaim 2, or a pharmaceutically acceptable salt thereof, wherein R² is


4. A compound of claim 2, or a pharmaceutically acceptable salt thereof,wherein R² is


5. A compound of claim 4, or a pharmaceutically acceptable salt thereof,wherein Z is Z is —O(CH₂)Ar, wherein Ar is pyridinyl optionallysubstituted with one substituent selected from C₁-C₄alkoxycarbonyl,C₁-C₄alkyl, amido, carboxy, cyano, and formyl.
 6. A compound selectedfrom1,1′-(2,8-diazaspiro[5.5]undecane-2,8-diyl)bis(4-((R)-3-(benzyloxy)pyrrolidin-1-yl)butan-1-one);1,1′-(2,8-diazaspiro[5.5]undecane-2,8-diyl)bis(4-((R)-3-hydroxypyrrolidin-1-yl)butan-1-one);(2S)-2-((4-(3-(8-(4-((R)-3-(benzyloxy)pyrrolidin-1-yl)butanoyl)-2,8-diazaspiro[5.5]undecan-2-yl)-3-oxopropyl)-2-((5-carbamoylpyridin-3-yl)methoxy)-5-chlorobenzyl)amino)-3-hydroxy-2-methylpropanoicacid;(2S)-2-((4-(3-(8-(4-((R)-3-(benzyloxy)pyrrolidin-1-yl)butanoyl)-2,8-diazaspiro[5.5]undecan-2-yl)-3-oxopropyl)-5-chloro-2-((5-cyanopyridin-3-yl)methoxy)benzyl)amino)-3-hydroxy-2-methylpropanoicacid; and(2S)-2-((4-(3-(8-(4-((R)-3-(benzyloxy)pyrrolidin-1-yl)butanoyl)-2,8-diazaspiro[5.5]undecan-2-yl)-3-oxopropyl)-5-chloro-2-((5-(methoxycarbonyl)pyridin-3-yl)methoxy)benzyl)amino)-3-hydroxy-2-methylpropanoicacid; or a pharmaceutically acceptable salt thereof.
 7. A pharmaceuticalcomposition comprising a compound of claim 1, or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier.
 8. Amethod of enhancing, stimulating, and/or increasing the immune responsein a subject in need thereof, said method comprising administering tothe subject a therapeutically effective amount of a compound of claim 1or a therapeutically acceptable salt thereof.
 9. A method of inhibitinggrowth, proliferation, or metastasis of cancer cells in a subject inneed thereof, said method comprising administering to the subject atherapeutically effective amount a compound of claim 1 or atherapeutically acceptable salt thereof.
 10. A method of treating aninfectious disease in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of acompound of claim 1 or a therapeutically acceptable salt thereof. 11.The method of claim 10 wherein the infectious disease is caused by avirus.
 12. A method of treating septic shock in a subject in needthereof, the method comprising administering to the subject atherapeutically effective amount of a compound of claim 1 or atherapeutically acceptable salt thereof.
 13. A method of blocking theinteraction of PD-L1 with PD-1 and/or CD80 in a subject, said methodcomprising administering to the subject a therapeutically effectiveamount of a compound of claim 1 or a therapeutically acceptable saltthereof.